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The iron responsive regulator Irr is found in a wide range of a-proteobacteria, where it regulates many genes in response to the essential but toxic metal iron. Unlike Fur, the transcriptional regulator that is used for iron homeostasis by almost all other bacterial lineages, Irr does not sense Fe2+ directly, but, rather, interacts with a physiologically important form of iron, namely heme. Recent studies of Irr from the N2-fixing symbiont Rhizobium leguminosarum (IrrRl) showed that it binds heme with submicromolar affinity at a His-Xxx-His (HxH) motif. This caused the protein to dissociate from its cognate DNA regulatory iron control element box sequences, thus allowing expression of its target genes under iron-replete conditions. In the present study, we report new insights into the mechanisms and consequences of heme binding to Irr. In addition to the HxH motif, Irr binds heme at a second, lower-affinity site. Spectroscopic studies of wild-type Irr and His variants show that His46 and probably His66 are involved in coordinating heme in a low-spin state at this second site. By contrast to the well-studied Irr from Bradyrhizobium japonicum, neither heme site of IrrRl stabilizes ferrous heme. Furthermore, we show that heme-free IrrRl exists as a mixture of dimeric and larger, likely hexameric, forms and that heme binding promotes IrrRl oligomerization. Bioanalytical studies of IrrRl variants showed that this property is not dependent on the HxH motif but is associated with heme binding at the second site.